This invention provides a reference electrode device of the constrained-diffusion liquid junction type useful in pH and/or ion-selective electrode (ISE) potentiometric sensors and is particularly suitable for use in a mini-integrated electrochemical analyzer.
Conventional types of reference electrodes have a liquid junction where the sample meets the junction solution. The junction is typically either open or constrained. In an open junction system, the liquid junction operates by free diffusion. In a constrained-diffusion junction system, a region of porous material permeable to water and salts (a membrane, porous plug, frit, or the like) is placed at the site of the liquid junction. The porous material acts as a constraint whereby passage of large molecules (such as protein) and bulk liquid transport is generally hindered.
The liquid junction solution (also commonly referred to as the salt bridge solution) typically contains a solution saturated with a salt (such as an equitransferent salt, including KCl, KNO3) which functions to reduce and maintain constant the interfacial potential which develops across the liquid junction boundary, typically referred to as a liquid junction potential. The difference in liquid junction potentials between the system calibrator and the sample is referred to as the residual liquid junction potential. Typically, the residual liquid junction potential increases as the ionic strength difference between the system calibrator solution and the sample solution increases. The residual liquid junction potential is generally considered to compromise the accuracy of the associated potentiometric sensors and therefore a multi-use reference electrode is typically designed to minimize the residual liquid junction potential for as long as possible, while balancing other design constraints.
In potentiometric systems that are designed with miniaturized working or indicator electrodes (typically pH and/or ion selective electrodes), the necessity of the junction solution makes miniaturization of a reference electrode difficult. Further, for the reference electrode to have a multiple use capability, the liquid junction solution must be present in a volume and concentration sufficient to minimize the residual liquid junction potential over its useful lifetime. This requisite volume particularly complicates miniaturization of constrained diffusion liquid junctions since the volume must typically be maintained close to the constraint to minimize problems associated with excessive ion depletion through the constraint. Another drawback of this type of reference electrode is that it tends to be orientation dependent. During operation, an upright orientation generally must be maintained with the liquid junction solution on top of the constraint in order to maintain contact therebetween. Moreover, in order to minimize errors due to the residual liquid junction potential, the junction solution is generally saturated with the equitransferent salt.
Conventional reference electrodes utilize a reference contact region (also sometimes referred to as electrode elements) immersed in a stagnant portion of the junction solution which contains a constant concentration of the equitransferent salt. The reference contact region is often silver based, consisting of an electrochemically reversible redox electrode couple such as Ag/Ag+ and Ag/AgCl. When salt solutions are used with silver based reference contact regions such as Ag/AgCl, the AgCl is susceptible to dissolution. In constrained-diffusion type liquid junctions, this dissolution is problematic because it leads to subsequent precipitation of silver salts on the region of porous material constraint, thus leading to undesirable fouling of the constraint, which in turn generally results in an erratic reference electrode performance.
Commonly, because of the above-described fouling problem associated with the use of silver based reference contact regions, barrier membranes have been used to restrict Ag+ ion migration from the reference contact region to the porous material constraint region. The use of a barrier membrane, however, carries with it inconvenience because the first use wet-up of the reference electrode is hindered by the barrier membrane, thus requiring a long soaking time in the junction solution prior to the first use. In addition, any additional component, such as this barrier membrane, tends to complicate miniaturization of the reference electrode.
Another common problem associated with the use of saturated equitransferent salt solutions in constrained-diffusion type of liquid junction reference electrodes is that when the reference electrode is stored and/or used at sub-ambient temperatures, salt crystallization and precipitation may occur between the reference contact region and the region of the porous material at the junction, which in turn leads to erratic reference electrode potentials. An additional problem associated with the use of a saturated equitransferent salt solution is that the saturated solution may contribute to reproducibility and/or accuracy problems with blood samples because of interference caused by precipitation of protein and crenation of red blood cells present in the sample.
Some of the aforementioned drawbacks associated with constrained diffusion liquid junctions are reduced or absent in open (free diffusion) liquid junctions. In this regard, reservoirs for holding of junction solutions have been described for open junction type reference electrodes, where there is no region of porous material to act as a constraint at the junction. For example, A. K. Covington et al. (Anal. Chim. Acta, 1985, 169, pp. 221-229) describe a open junction where the junction solution is moved from a KCl reservoir via a syringe. In this prior art system, the liquid junction is established with each sample, but because this type of system leads to cross-contamination of the liquids upon use, the junction solution must be discarded along with the sample thus leading to increased waste. Moreover, such contamination tends to dictate placement of the reference electrode downstream in the sample path of all working electrodes. This restriction disadvantageously limits flexibility of analyzer configuration.
Moreover, open free-diffusion liquid junction reference electrodes tend to require specific orientations and geometries to provide good reproducible junctions. Reproducibility for open free-diffusion liquid junctions tends to depend on uniform, generally circular junction geometries and a constant orientation. (See, e.g., R. E. Dohner et al. Anal. Chem., 1986, 58, pp. 2585-2589; T. R. Harbinson et al., Anal. Chem., 1987, 59, pp. 2450-2456). The orientation and geometries requirements are particularly limiting when attempting to adapt such a reference electrode to a miniaturized, modular system in which it may be desirable to locate the reference electrode within an array of working electrodes and utilize componentry and dimensions common to those working electrodes. The aforementioned orientation requirement may be particularly troublesome in the event a miniature portable analyzer is desired for use in locations where the electrode array may be utilized in various or unstable orientations, such as may be encountered in the field or in mobile applications such as on board ambulances, ships and/or aircraft.
Thus, although many different reference electrodes are known in the prior art, there is a need to discover alternative reference electrodes for electrochemical analyzers, particularly modular reference electrodes that may be easily adapted for use in a mini-integrated type of analyzer.
Many of the shortcoming associated with prior art reference electrodes have been overcome with the discovery of a reference electrode assembly of the present invention.
According to the invention, provided is a modular reference electrode assembly adapted for serial integration within an orientation independent array of working electrodes, the working electrodes disposed in a plurality of electrode receiving positions on a support member of an integrated sample analyzer. The reference electrode includes:
a flow cell of modular construction sized and shaped for selective disposition in any of the plurality of electrode receiving positions on the support member, wherein said flow cell is adapted for being serially retained within a sample flow path of the array;
a flow cell of modular construction sized and shaped to interfit between guide members adapted to retain the array of miniaturized working electrodes, wherein the flow cell is adapted for being serially retained within a sample flow path of the array;
a liquid junction disposed within the flow cell;
a remote reservoir for holding a liquid junction solution, the remote reservoir being connected to the flow cell by a liquid junction flow path, the liquid junction flow path being separate from the sample flow path;
a reference contact region in physical contact with the liquid junction solution;
the liquid junction including a constraint having a region of porous material permeable to water and salts, the constraint having a contact portion adapted for contacting the liquid junction solution on one side thereof and a sample solution on an other side thereof;
the constraint adapted to substantially prevent bulk flow of the liquid junction solution therethrough, and to provide an orientation independent, stable liquid junction; and
means for moving the liquid junction solution from the reservoir to the constraint and for applying pressure sufficient to maintain the liquid junction solution in contact with the constraint at substantially any orientation of the array of electrodes wherein the flow cell is operable at substantially any orientation.
Also provided is a method for providing a modular, orientation independent reference electrode for a system for potentiometric quantitative analysis. This method includes:
providing a flow cell of modular construction sized and shaped for selective disposition in any of a plurality of electrode receiving positions on a sensor support member adapted to support an array of working electrodes, wherein the flow cell is adapted for being serially retained within a sample flow path of the array;
interfitting the flow cell in series with the array of miniaturized working electrodes;
moving a sample solution through the sample flow path over at least one miniaturized working electrode disposed within the array;
moving the sample solution to the flow cell to form a liquid junction between the sample solution and a liquid junction solution over a porous constraint wherein the sample solution is disposed in contact with one side of the porous constraint and the liquid junction solution is disposed in contact with an other side of the porous constraint, the constraint being adapted to substantially prevent bulk flow of the liquid junction solution therethrough and to provide an orientation independent, stable liquid junction, the liquid junction solution comprising a non-saturated equitransferent salt and being stored in a remote reservoir wherein the liquid junction solution is pumped to the constraint per sample cycle and the liquid junction solution is electrically connected with sensing equipment by a reference contact region in physical contact with the liquid junction solution;
maintaining the liquid junction solution in contact with the constraint with sufficient pressure wherein the flow cell is operable at substantially any orientation of the array of electrodes; and
measuring an electric potential developed between the reference contact region and the working electrode.
The inventive reference electrode assembly is particularly useful because it is not orientation and gravity specific. The modular construction enables it to be interchangeably retained at substantially any predetermined location within an array of working electrodes in a configuration similar to that utilized for the working electrodes. Moreover, the present invention may be fabricated as a multiple use reference electrode for use in a mini-integrated electrochemical analyzer having planar miniaturized working electrodes because sufficient liquid junction solution is present for multiple uses without presenting a space problem since the extra liquid junction solution is stored in the reservoir that is remote from where the constrained-diffusion liquid junction is created. Other advantages of preferred embodiments are described herein.